Fluorescent nanoparticles have been found useful as visualization tools for biological sensing, probing, imaging, and monitoring. The development of fluorescent probes for biomolecular detection has emerged as an exciting area of research because of its importance in bioscience and biotechnological applications, as well as its impact on public health. The fluorescent assay process offers a number of advantages over other analytical techniques, such as rapid response, high sensitivity, low background noise, and wide dynamic working range. Thanks to the enthusiastic effort of scientists devoted to this area of research, a large variety of fluorescent bioprobes have been developed. However, many of the bioprobes work in a “turn off” mode. For example, the emission of a fluorophore is switched “off” when it interacts with a quenching species in a biological system through a mechanism of fluorescence resonance energy transfer.
Typical materials used as biosensors include natural polymers, inorganic nanoparticles, and organic dyes. Green fluorescent protein (GFP), for example, has been used as a reporter of expression for morphological differentiation. The biosensing process, however, requires complicated and time-consuming transfection procedures, which can lead to unexpected morphologies and undesired abnormality in the target cells. Inorganic nanoparticles, such as semiconductor quantum dots (QDs), are highly luminescent and resistant to photobleaching but limited in variety and inherently toxic to living cells because QDs are commonly made of heavy metals and chalcogens (e.g., CdS, CdSe, CdTe, PbS, and PbSe).
Among the nanoparticles, QDs have attracted a lot of attention, particularly in the area of cellular marking and imaging. QDs enjoy such advantages as size-tunable emission color, long luminescence lifetime, and resistance to photobleaching. However, QDs are limited in variety, difficult to access, chemically unstable in harsh environments, difficult to dispose of, and highly cytotoxic to living cells because they are commonly made of heavy metals and chalcogens (e.g., CdS, CdSe, CdTe, PbS, and PbSe). These limitations present challenges to scientists from academic to industrial sectors.
Organic dyes are rich in variety and have been widely used as readily processable light-emitting materials, particularly in the area of organic optoelectronics. Due to their poor miscibility with water, organic dyes are prone to aggregate in aqueous media, which normally weakens their light emissions. This effect is commonly known as aggregation-caused quenching (ACQ).
Alternatively, organic fluorophores, such as fluorescein and rhodamine, have been used. Thanks to the elaborate efforts of various scientists, a wide variety of luminogenic materials covering a wide range of absorption and emission wavelengths have been prepared and specialized for particular applications. However, when these fluorophores are worked into acidic or basic media with enzymes and ions, their emissions are quenched through multiple nonradiative pathways.
For sensitive detection, trace analysis, diagnostic assays, and real-time monitoring, fluorescent bioprobes must emit intense visible light upon photoexcitation. However, light emissions from most fluorophores are rather weak. This aggregation-caused quenching (ACQ) is due to emission quenching caused by the aggregation of fluorophores in the solid state. When dispersed in aqueous media or bound to biomolecules, fluorophore molecules are inclined to aggregate, which usually quenches their fluorescence, and thus, greatly limits their effectiveness as bioprobes. The ACQ effect also makes it difficult to assay low-abundance molecular species in biological systems because the fluorescence signals from minimal amounts of fluorophores matching the bioanalyte levels may be too weak to be determined accurately. In addition, at high fluorophore concentrations, the emissions are further weakened, rather than enhanced, due to the ACQ effect.
Accordingly, there is a great need for the development of fluorescent bioprobes for bioimaging that are resistant to the ACQ effect. Furthermore, the fluorescent bioprobes must have high biological compatibility, strong photobleaching resistance, efficient light emission, high selectivity and sensitivity, and must be nontoxic to living cells.